LECTURE 11

PROTOPLAST FUSION AND SOMATIC HYBRIDIZATION

Protoplast fusion is especially important in sexually incompatible plants and in cases
where conventional methods of breeding fails to operated.
Protoplast fusion can be classified into three categories.1. Spontaneous fusion, 2. Mechanical
Fusion 3.Induced fusion.
Spontaneous fusion
Young leaves are more likely to produce such spontaneously induced multi nucelate protoplasts. It
is strictly inter specific.
Mechanical fusion
This is a mechanical method to bring isolated protoplasts into intimate contact through micro
manipulators and perfusion micropipette. By this method, occasional fusions of protoplasts from
soybean. Arachis hypogea and Vinca rosea were observed. In this procedure,
protoplasts are likely to get injury.
Induced fusion
Induced fusion of protoplasts does not necessarily involve fusion of the same plant species which
requires an inducing agent.
Use of fusogens
1. Treatment with NaNO3: Isolated protoplasts are suspended in an aggregation mixture of
5.5% Sodium nitrate in 10% sucrose solution
2. Effect of proteins: Gelatin and early products of its degradation at a concentration of 2.5%
induced aggregation at high frequency within one hour E.g. Vicia, Glycine and Allium.
3. Immunological Method: Soybean and brome grass antibody cross reacted with and
agglutinated Vicia protoplasts..
4. Calcium ions at High pH: This involves spinning the protoplasts in a fusion inducing
solution (0.05M CaCl2 2H2O in 0.4 M mannitol at pH 10.5)
5. Polyethylene Glycol (PEG)
When protoplasts are available in sufficient quantities, 1 ml of the protoplasts suspended in a
culture medium is added to 1 ml of 56% solution of PEG and the tube shaken for 5 Seconds.
6.Electric fusion: There are two steps:
Protoplasts are exposed to high frequency alternating electric field (0.5-1.5 NH 2) that generates
dipoles through dielectricphores. So, protoplasts find together to form, pearl chains.
Application of one/more short (10-100s) direct current (DC) (1.3 k/cm -1). Cause reversible
membrane breakdown resulting in pores in the aligned membranes. The contact membranes can
be fused and fusion opens the way for hybrid cell formation. To insure maintenance of close
membrane contact during fusion, AC field is reapplied.

Selection of fused protoplasts

After fusion, the protoplast population consists of a mixture of parental types, homokaryons and
heterokaryons, of which heterokaryons (potential source of future hybrids) often make only 0.5%10%.
1. Visual selection: This utilizes protoplasts for fusion studies that are visually distinct at the light
microscope level. This is laborious. Only limited fusion products can only be selected.
2. Biochemical basis of selection/Nutrional selection:This is based on nutritional
requirements of parents and hybrids
3.Complementation Selection is based upon the ability of the two genomes present in the
heterokayon/hybrid to complement E.g. N. tabaccum Petunia Datura.
4. Drug resistance/sensitivity: Parental types are sensitive to actinomycin. (Petunia
parodic, P. hybrida). Somatic hybrids (P. hybrida + P. parodri) are resistant to
actinomycin.
5. Labelling: Protoplasts of two parents may be labeled by different fluorescent agents, which
will then enable the selection of hybrids
(1) Octadeconyl amino fluorscein.
(2) Octadecyl palamine.
6.Fluorescent Assorted cell sorter (FACS):
Uses
Somatic hybrids for gene transfer
- Interspecific hybrids in genus-Daucus and its relatives.
Rice and Echinochola oryzicola (Barnyard grass) hybrids have been obtained E.g.
Nicotinana, Brassica, Petunia, Solanum, Lycopersicon and Datura interspecific hybrids.
-Intergeneic
Raphanus sativus + B. oleracea Rhaphnobrassica
Moricandia arvensis + B. oleracea - Moricandio brassica
N. tabacum + L. esculentum - Nictoipersicon
Solanum tuberosum + L. esculentum - Solanopersican
Datura innoxia + Atropa belladona - Daturotropa.
O.sative + Echinochloa oryzicola-Oryzochloa.
Intertribal (bet. family, Brassicaceae)
A. thaliana + B. campestris - Arabidobrassica
Barbarea vulgaris + B. napus - Barbareobrassica
Symmetric hybrids between potato and tomato have been shown to exhibit intermediate
cold tolerance.
Somatic hybrids for CMS (Melchers, 1992).
Substitute nucleus of one species into the cytoplasm of another species, whose
mictochondria are inactivated.which led to generation of CMS.
E.g. Mesophyll protoplasts of Solanum acaule (potato) and tomato.
In five tomato cultivars male sterility induced in this manner was inherited maternally over
several generations.